INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, EMULSIFIERS, STABILIZERS,
ANTI-CAKING AGENTS AND CERTAIN
OTHER SUBSTANCES
FAO Nutrition Meetings Report Series
No. 46A WHO/FOOD ADD/70.36
The content of this document is the result of the deliberations of the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
27 May - 4 June 19691
Food and Agriculture Organization of the United Nations
World Health Organization
1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food
Additives, FAO Nutrition Meetings Report Series, in press;
Wld Hlth Org. techn. Rep. Ser., in press.
BRILLIANT BLUE FCF
Biological Data
Biochemical aspects
The colour was administered orally to rats as a two per cent. aqueous
solution at a level of 200 mg per rat. Almost the entire amount was
excreted unchanged in the faeces within 40 h after administration. In
a later investigation, the presence of the colour in the bile was
observed in rats, rabbits and dogs after oral administration. In the
case of the dog, the amount did not exceed five per cent. of the dose
administered (Hess & Fitzhugh, 1953; 1954; 1955). Administration of an
aqueous solution of the colour by stomach tube resulted in 89 per
cent. excretion in the faeces; none was found in the urine. After
subcutaneous injection of 80-100 mg some 79 per cent. was excreted; 77
per cent. appeared in the faeces and 2.5 per cent. in the urine
(Imperial Chemical Industries, 1958).
Acute toxicity
Animal Route LD50 Reference
mg/kg body weight
Mouse Subcutaneous 4 600 Gross, 1961
Rat Oral > 2 000 Lu & Lavallée, 1964
Groups of five young rats were given injections of the colour
subcutaneously twice daily for three days. The rats were killed on the
fourth day. The colour was administered in aqueous solution at a level
of 250 mg/kg body weight each injection. No oestrogenic activity was
detected. No other abnormalities were found (Graham & Allmark, 1959),
Special studies
o-Sulfobenzaldehyde, one of the components of Brilliant Blue FCF, was
fed to groups of three-week-old rats, 10 males and 10 females per
group, at levels of 0, 0.25, 0.5, 1 and 2 per cent. for a period of 13
weeks followed by gross and histopathology examinations. The
"no-effect" level of o-sulfobenzaldehyde was considered to be two per
cent. (United States Food and Drug Administration, 1969).
Short-term studies
Mouse: Daily injections of 2 mg of colour over 30 days were well
tolerated by mice. Some animals showed swelling of the liver and
spleen after receiving a total of 4 mg (Gross, 1961). Mice fed 1200 mg
of the dye ever 19 days showed no damage (Gross, 1961).
Rat: Subcutaneous injection of 1 ml of 0.8 per cent. aqueous
solution twice weekly produced histological changes suggestive of
subsequent sarcoma formation unassociated with chemical carcinogenic
potential (Grasso & Golberg, 1966).
Dog: Studies were conducted on beagle dogs fed diets containing one
per cent. and two per cent. of the colour for one year. No gross or
microscopic changes due to treatment were seen (Hansen et al., 1966).
Cat: Parenteral injection into cats of a five per cent. aqueous
solution, 1.0 g on the first day and 0.1 g from the ninth to the
eighteenth day, did not produce methaemoglobinaemia or Heinz bodies
(Gross, 1965).
Mouse: A group of 57 male and 43 female mice were given 1 mg colour
per day in the diet. Observations extended over 500-700 days. No
evidence of carcinogenic action was found (Waterman & Lignac, 1958).
Subcutaneous injections of 10 doses of 4 mg followed by 50 doses of 6
mg showed no tumour production after 78 weeks (Imperial Chemical
Industries, 1962).
Rat: The colour was fed in a concentration of four per cent. of the
diet to five male and five female rats for 600 days. Gross staining of
the glandular stomach and some granular deposits in the stomach but no
tumours were observed (Willheim & Ivy, 1953).
Eighty-five rats were fed a diet containing 0.1 per cent. of the
colour for their life span. The daily intake was 10-15 mg. No tumours
were found (Klinke, 1955). Groups of 24 weanling Osborne-Mendel rats,
evenly divided by sex, were fed the colour at levels of 0, 0.5 per
cent., 1.0 per cent., 2.0 per cent. and 5.0 per cent. for two years.
No outward effects were observed during the experiment. Gross and
microscopic examination revealed only incidental lesions, unrelated to
treatment, with no effect on tumour production. The statistical
evaluation revealed no changes in organ weights, mortality, or growth.
Haematology was normal (Hansen et al., 1964). Four groups of 15 male
and 15 female rats were given diets containing, 0, 0.03 per cent., 0.3
per cent. and 3.0 per cent. of the colour for 75 weeks. No depression
of growth, food consumption or food efficiency was found. Mortality
was increased in the three per cent. group due to respiratory
infection unrelated to the administration of the compound.
Haematological findings were normal (Mannell et al., 1962).
Subcutaneous injection of various preparations of the colour have been
reported to produce fibrosarcomas at the site of the injection (Nelson
& Hagan. 1953; Gross, 1961; Hansen et al., 1966). However, no tumours
were produced by a course of subcutaneous injections of 0.5 ml of a
four per cent. solution in isotonic saline (Mannell & Grice, 1964).
Eighty-four male and 84 female Wistar rats were given twice-weekly 10
doses of 20 mg per rat as a two per cent. solution followed by 50
doses of 30g per rat as a six per cent, solution. One hundred and
nineteen of the 168 rats developed sarcomas at the site of injection.
Only seven animals survived two years. Males had no other tumours but
females had six mammary, one ovarian, one uterine tumour and two
hepatomas. Twenty-six controls survived two years out of 48 and the
females developed one mammary, one ovarian and four uterine
tumours (Imperial Chemical Industries, 1962). Six male and six female
Slonacker rats were treated the same way as Wistar rats. All died
within 420 days, tumours developing at the injection site in five
males and three females. Out of 12 controls, 10 were alive at 400
days, No tumours developed in controls (Imperial Chemical Industries,
1962).
Comments
The production of a high percentage of local sarcomata at the site of
subcutaneous injection in rats has led in the past to considerable
discussion and consequently to extensive studies on this colour. The
production of these sarcomata is considered to be related to the
physico-chemical properties of the colour and special condition of the
experiment and does not constitute evidence of carcinogenicity by the
oral route.
Biochemical studies have shown that the colour is poorly absorbed and
is almost completely excreted in the faeces after parenteral
administration. Extensive long-term studies in two species are
available. In addition a 13-week study in rats with
o-sulfobenzaldehyde, one of the components of commercial Brilliant
Blue FCF, has been carried out. Oral feeding produced no pathological
changes at the highest levels used in adequate experiments.
Biochemical studies on metabolism using modern techniques are
desirable.
EVALUATION
Level causing no toxicological effect in the rat
Five per cent, in the diet (= 50 000 ppm) equivalent to 2500 mg/kg
body weight/day.
Estimate of acceptable daily intake for man
mg/kg body weight/day
Unconditional acceptance
0-12.5
REFERENCES
Graham, R. C. B. & Allmark, M. G. (1959) Toxic. Appl.Pharmac., 1,
144
Grasso, P. & GoIberg L. (1966) Fd. Cosmet. Toxicol., 4, 269
Gross, E. (1961) Z. Krebsforsch., 64, 287
Hansen, W. H. et al. (1966) Toxicol. Appl. Pharmacol., 8, 29
Hess, S. M. & Fitzhugh, O. G. (1953) Fed. Proc., 12, 330
Hess, S. M. & Fitzhugh, O. G. (1954) Fed. Proc., 13, 365
Hess, S. M. & Fitzhugh, O. G. (1955) J. Pharmacol. exp. Ther., 114,
38
Imperial Chemical Industries (1958) Unpublished report
Imperial Chemical Industries (1962) Unpublished report
Lu, F. C. & Lavallée., A. (1964) Canad. pharm. J., 97, 30
Mannell, W. A., Grice, H. C. & Allmark, M. G. (1962) J. Pharm.
Pharmacol., 14, 378
Mannell, W. A. & Grice, H. C. (1964) J. Pharm. Pharmacol., 16, 56
Nelson, A. A. & Hagan, E. C. (1953) Fed. Proc., 12, 397
United States Food and Drug Administration (1969) Unpublished report
Willheim, R. & Ivy, A. C. (1953) Gastroenterology, 23, 1
Waterman N. & Lignac, G. O. E. (1958) Acta. physiol. pharmacol.
neerl., 7, 35